Background: Mesenchymal stem cells are heterogenous populations with hematopoietic supporting and immunomodulating capacities. Enormous studies have focused on their preclinical or clinical therapeutic effects, yet the systematic study of continuous in vitro passages on signatures and functions of UC-MSCs at both the cellular and molecular levels is still lacking. Methods: In this study, to systematically evaluate the biological properties of MSCs at various passages, we analyzed biomarker expression, cell proliferation and apoptosis, chromosome karyotype, and tri-lineage differentiation potential. Subsequently, we took advantage of whole-exome sequencing to compare the somatic hypermutation of hUC-MSCs at P3, P6, and P15 including SNV and INDEL mutations. In addition, to explore the safety of the abovementioned hUC-MSCs, we performed metabolic pathway enrichment analysis and in vivo transplantation analysis. Furthermore, we cocultured the abovementioned hUC-MSCs with UCB-CD34 + HSCs to evaluate their hematopoietic supporting capacity in vitro. Finally, we transplanted the cells into acute graft-versushost disease (aGVHD) mice to further evaluate their therapeutic effect in vivo. Results: The hUC-MSCs at P3, P6, and P15 showed similar morphology, biomarker expression, and cytokine secretion. hUC-MSCs at P15 had advantages on adipogenic differentiation and some cytokine secretion such as IL-6 and VEGF, with disadvantages on cell proliferation, apoptosis, and osteogenic and chondrogenic differentiation potential. Based on the SNP data of 334,378 exons and bioinformatic analyses, we found the somatic point mutations could be divided into 96 subsets and formed 30 kinds of signatures but did not show correlation with risk of tumorigenesis, which was confirmed by the in vivo transplantation experiments. However, hUC-MSCs at P15 showed impaired hematologic supporting effect in vitro and declined therapeutic effect on aGVHD in vivo.

Objective Longitudinal studies have indicated VCAM‐1+ mesenchymal stem/stromal cells (MSCs) as promising resources in regenerative medicine, yet the abundance in gene expression is far from adequate in the advantaged and “discarded” hUC‐MSCs. Thus, high‐efficient preparation and systematic dissection of the signatures and biofunctions of the subpopulation is the prerequisite for large‐scale clinical applications. Materials and methods We primarily took advantage of a cytokine‐based programming strategy for large‐scale VCAM‐1+ hUC‐MSC generation (III‐MSCs). Thereafter, we conducted multifaceted analyses including cytomorphology, immunophenotype, cell vitality, multilineage differentiation, whole‐genome analysis, tube formation and Matrigel plug assay, lymphocyte activation and differentiation, and systemic transplantation for aplastic anaemia (AA) treatment. Results III‐MSCs with high‐proportioned VCAM‐1 expression were obtained by combining IL‐1β, IL‐4 with IFN‐γ, which exhibited comparable immunophenotype with untreated hUC‐MSCs (NT‐MSCs) but revealed multidimensional superiorities both at the cellular and molecular levels. Simultaneously, systemic infusion of III‐MSCs could significantly ameliorate clinicopathological features and finally help facilitate haematopoietic reconstruction and immunoregulation in AA mice. Conclusions We have established a high‐efficient procedure for large‐scale generation of III‐MSCs with preferable signatures and efficacy upon aplastic anaemia in mice. Our findings suggested that III‐MSCs were advantageous sources with multifaceted characteristics for regenerative medicine.

Background: Mesenchymal stem/stromal cells (MSCs) derived from human embryonic stem cells (hESCs) are attractive for their hematopoietic-supporting or potential therapeutic effects. However, procedures for high-effective and scalable generation of MSCs from hESCs within 2 weeks are still unestablished, which also hinder the development and mechanism study of mesengenesis. Methods: In this study, we aimed to establish a strategy for programming hESC differentiation into MSCs by practicing small-scale chemical compound screening. Then, we used flow cytometry, multi-lineage differentiation, and karyotype analyses to investigate the biological phenotypes of the derived hESC-MSCs. Also, to explore whether the derived cells had hematopoietic-supporting ability in vitro, we carried out the cobblestone formation and megakaryocytic differentiation experiments. To further evaluate the function of hESC-MSCs in vivo, we transplanted the cells into a mouse model with hind limb ischemia. Results: By simultaneous treatments with a JAK/STAT antagonist and a DNA methylation inhibitor, the efficiency of generating hESCs into CD73 + hESC-MPCs could reach 60% within 7 days. The derived cells further matured into hESC-MSCs, with comparable characteristics to those of adult MSCs in terms of surface markers, normal karyotype, and the potential for adipogenic, osteogenic, and chondrogenic differentiation. Functionally, hESC-MSCs had hematopoieticsupporting effects in vitro and could notably relieve symptoms of hind limb ischemia. Conclusions: In the study, we established a high-efficient procedure for large-scale generation of MSCs from hESCs, which would be of great help for genesis and mechanism studies of MSCs. Meanwhile, the derived cells provide an alternative for translational clinical research.